(1) Field of the Invention
The present invention relates to a process for the preparation of novel DNases which involves culturing a novel transformant belonging to the genus Bacillus and collecting simultaneously at least two or more DNases possessing different substrate specificity, that is, the enzymes property of recognizing a specific nucleotide sequence of deoxyribonucleic acid and cleaving its bonds to produce unique fragments of the deoxyribonucleic acid.
(2) Description of the Prior Art
Enzymes (DNase) which decompose deoxyribonucleic acid (DNA), exist in various living things, and participate in important life processes, such as metabolism, decomposition, synthesis and recombination. Accordingly characteristics of enzyme particularly, in the field of protein chemistry and biological functions of these enzymes have recently attracted attention in the art.
In the study of the structure and function of the genes, it is important to produce and separate enzymes having specific actions, and deoxyribonucleic acid decomposing enzymes are valuable as biochemical reagents for molecular cloning of the genes and breed improvement.
DNases are roughly classified into two types, namely, exonucleases and endonuclease, according to their mode of action. More specifically, the former enzyme acts on a polynucleotide from its terminal and successively liberates nucleotide or oligonucleotide. The latter enzyme cleaves phosphodiester bonds in the DNA molecule.
In the field of endonuclease type enzymes, great progress has recently been made in the studies on enzymes showing a specificity of the structure of DNA particularly of the configuration of nucleotide or to the structural change which exists in nature or is introduced artificially, enzymes capable of recognizing structure of molecule and acting thereon and enzymes having a biologically important function (see Tadahiko Ando, Chemistry and Life, 13, 6, p. 342, 1975).
The present inventors have carried out research on enzymes and already established a process for production of an enzyme, which does not act on double-stranded DNA but specifically decomposes single-stranded DNA, from a culture broth of Aspergillus oryzae (see Japanese Pat. No. 593,368), a process for producing an enzyme capable of preferentially cleaving a purine-purine linkage in the DNA molecule, from cells of Aspergillus oryzae (see Japanese Pat. No. 621,205), a process for producing an enzyme capable of introducing single-strand breaks in DNA from bacteriophage-infected Escherichia coli (see Japanese Pat. No. 764,919), a process for producing an enzyme capable of acting on RNA and giving nucleoside cyclic phosphate from Escherichia coli by infection or induction with bacteriophage (see Japanese Pat. No. 829,334), a process for producing an enzyme capable of preferentially cleaving a guanine-guanine linkage in the DNA molecule from a culture broth of an alkalophilic bacteria (see Japanese Pat. No. 831,171), a process for producing from cells of Bacillus subtilis, an enzyme capable of specifically decomposing RNA moiety of DNA-RNA hybrid (see Japanese Pat. No. 877,668), and a process for producing an enzyme from Bacillus subtilis or and related strains belonging to the genus Bacillus having a substrate specificity to DNA: this enzyme recognises and cleaves specific nucleotide sequence (see Japanese Patent Application No. 121671/75 (Laid-Open Specification No. 47980/77) and Japanese Patent Application No. 87883/76 (Laid-Open Specification No. 15491/78)).
Microorganisms have a capability of specifically recognizing and decomposing foreign DNA in the cell (restriction system) and also have a capability of modifying such DNA so that the DNA shows a resistance to its own restriction system (modification system).
The restriction enzyme is an endonuclease participating in this restriction-modification system, and the restriction enzyme includes an enzyme capable of recognizing a specific sequence of 4 to 6 nucleotides in the molecule of a double-standard DNA and cleaving the sequence or in the vicinity of the sequence.
By virtue of such substrate specificity, these restriction enzymes can be effectively utilized for the studies on analysis of primary structure of DNA (nucleotide sequence) and its function and on in vitro recombination of heterogenous DNA.
The present invention provides an industrial process for the production of these restriction enzymes which are valuable as biochemical reagents.
The present inventors have carried out research on production, separation and substrate specificity of restriction enzymes prepared from microorganisms belonging to the genus Bacillus. We have succeeded in developing a process in which Bacillus subtilis marburg 168 (GSY 1026) as a recipient is subjected to transformation with DNA of Bacillus subtilis R to introduce the restriction-modification system of the strain R into the recipient, the resulting transformant ISMR 4 is selected, the selected transformant ISMR 4 as a recipient is subjected to transformation with DNA of Bacillus subtilis IAM 1247 to introduce the restriction-modification system of the strain 1247 into the recipient, the resulting transformant ISMRB 9 is selected, the selected transformant ISMRE 9 as a recipient is similarly subjected to transformation with DNA of Bacillus subtilis IAM 1231 to introduce the restriction-modification system of the strain 1231, and the resulting transformants ISMRBE 17 and ISMBF 21 are selected.
The above-mentioned transformant ISMRB 9 has restriction-modification systems of the strains GSY 1026, R and IAM 1247 in combination. We further succeeded in developing a process in which the transformant ISMRB 9 is cultured and a restriction enzyme Bsu R inhered in the strain R and a restriction enzyme Bsu 1247I inhered in the strain IAM 1247 are simultaneously prepared from cells of the transformant ISMRB 9.
Similarly, it has been confirmed that the transformant ISMRBE 17 has restriction-modification systems of the strains GSY 1026, R and IAM 1247 and a second restriction-modification system of the strain IAM 1231 in comibination, and that the transformant ISMBR has restriction-modification systems of the strains GSY 1026 and IAM 1247 and a second restriction-modification system of the strain IAM 1247.
Based on the foregoing findings, we have succeeded in simultaneously preparing three enzymes, that is, restriction enzymes Bsu R, Bsu 1247I and Bsu 1231 I inhered in the strains R, 1247 and 1231, respectively, by culturing the above-mentioned transformant ISMRBE 17.
We have also succeeded simultaneously preparing restriction enzymes Bsu 1247I and Bsu 1231II inhered in the strains IAM 1247 and IAM 1231 by culturing the above-mentioned transformant ISMBF 21.
Similarly, we have succeeded in developing a process in which the strain GSY 1012 derived from the above-mentioned strain GSY 1026 is used as a recipient and subjected to transformation with DNA of the above-mentioned strain IAM 1231 to introduce the restriction-modification systems of the strain IAM 1231, the resulting transformants ISE 15 and ISF 18 then being selected.
The transformant ISE 15 has a first restriction-modification system inhered in the strain IAM 1231. We have succeeded in preparing a restriction enzyme Bsu 1231I inhered in the strain IAM 1231 from cells obtained by culturing the transformant ISE 15.
The transformant ISF 18 has a second restriction-modification system inhered in the strain IAM 1231. We have succeeded in preparing a restriction enzyme Bsu 1231 II inhered in the strain IAM 1231 from cells obtained by culturing the transformant ISF 18.
Similarly, we have succeeded in developing a process in which the strain GSY 1012 derived from the strain GSY 1026 is used as a recripient and subjected to transformation with DNA of the abovementioned strain IAM 1247 to introduce the restrictionmodification system of the strain IAM 1247 into the recipient, the resulting transformant ISB 8 then being selected. This transformant ISB 8 has a restriction-modification system of the strain IAM 1247.
We have succeeded in preparing a restriction enzyme Bsu 1247I inhered in the strain IAM 1247 from cells obtained by culturing the above-mentioned transformant ISB 8.
We have now completed the present invention based on the foregoing achievements.